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Xu F.,China Agricultural University | Wang W.,Beijing WDWK Biotechnology Corporation | Jiang H.,China Agricultural University | Wang Z.,Beijing WDWK Biotechnology Corporation | And 2 more authors.
Food Analytical Methods | Year: 2014

Plasticizer has attracted more and more attention in China for the past 3 years, especially in Taiwan district. In this study, an indirect competitive enzyme-linked immunosorbent assay (icELISA) has been developed for the determination of a plasticizer dibutyl phthalate (DBP) in white wine. Dibutyl 4-aminophthalate coupled with OVA was synthesized as an immunogen to produce polyclonal antibodies against DBP. The antibody exhibited negligible cross-reactivity with other related compounds. The influence of several physicochemical parameters, such as coating procedure, organic solvent, competitive reaction time, and pH was investigated. The limit of detection was 64.5 ng/mL, which was sensitive enough for a screening assay. The linear range was 64.5-1,606.2 ng/mL with a correlation coefficient (R 2) of 0.996. The method was successfully applied to the determination of DBP in white wine. Recoveries were between 83.1 and 101.7 %. This immunoassay was highly specific, sensitive, rapid, simple, and suitable for DBP monitoring. The results obtained were compared with those obtained using gas chromatography-mass spectrometry (GC-MS), and a satisfied correlation coefficient of 0.928 was obtained by real sample detection. © 2014 Springer Science+Business Media New York.


Li X.,China Agricultural University | Li X.,Beijing WDWK Biotechnology Company | Wen K.,China Agricultural University | Chen Y.,China Agricultural University | And 5 more authors.
Food Analytical Methods | Year: 2015

A rapid, simple, reliable, and sensitive immunogold chromatographic assay (IGCA) was described for simultaneous determination of four macrolide antibiotics (erythromycin, spiramycin, tilmicosin, and tylosin) in raw milk. Three antigens were immobilized as three test lines on the nitrocellulose membrane, which enable the simultaneous determination on a single test strip. Samples were detected directly without treatment; the entire testing process was completed within 10 min. The visual detection limits for erythromycin, spiramycin, tilmicosin, and tylosin were 5, 5, 10, and 20 ng/mL, respectively. Sixty blind raw milk samples were analyzed by both IGCA and liquid chromatography–tandem mass spectrometry; the results showed a good correlation between the two methods. The results demonstrate that the developed method could provide a rapid and effective approach for the onsite determination of multi-macrolide antibiotics residues in a large number of samples. © 2015, Springer Science+Business Media New York.


Li X.,China Agricultural University | Luo P.,China National Institute for Nutrition and Food Safety | Tang S.,China Agricultural University | Beier R.C.,U.S. Department of Agriculture | And 4 more authors.
Journal of Agricultural and Food Chemistry | Year: 2011

A simple, rapid and sensitive immunogold chromatographic strip test based on a monoclonal antibody was developed for the detection of melamine (MEL) residues in raw milk, milk products and animal feed. The limit of detection was estimated to be 0.05 μg/mL in raw milk, since the detection test line on the strip test completely disappeared at this concentration. The limit of detection was 2 μg/mL (or 2 μg/g) for milk drinks, yogurt, condensed milk, cheese, and animal feed and 1 μg/g for milk powder. Sample pretreatment was simple and rapid, and the results can be obtained within 3-10 min. A parallel analysis of MEL in 52 blind raw milk samples conducted by gas chromatography-mass spectrometry showed comparable results to those obtained from the strip test. The results demonstrate that the developed method is suitable for the onsite determination of MEL residues in a large number of samples. © 2011 American Chemical Society.


Li X.,China Agricultural University | Li X.,Beijing WDWK Biotechnology Company | Wang W.,Beijing WDWK Biotechnology Company | Wang L.,China Agricultural University | And 3 more authors.
Analytical and Bioanalytical Chemistry | Year: 2015

Phenylethanolamine A (PA) is a β-adrenergic agonist, which was first used in animal husbandry as a growth promoter in China in 2010. In this study, a monoclonal-antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) and lateral-flow immunoassay (LFA) for the detection of PA in swine urine and pork were developed. The immunogen was prepared by linking PA hapten with carrier protein via a diazotization method. The IC50 value of the optimized icELISA was 0.44 ng mL−1. The limits of detection of the icELISA for PA in swine urine and pork were 0.13 ng mL−1 and 0.39 ng g−1, respectively. The recoveries of PA from spiked swine urine and pork were in the range 82.0–107.4 % and 81.8–113.3 %, respectively, with the coefficients of variation in the range 4.1–16.2 % and 1.2–6.3 %, respectively. The mAbs had negligible cross reactivity with 10 other β-agonists. In contrast, the LFA had a cut-off level of 5 ng mL−1 (g) in swine urine and pork, and the results could be achieved within 5 min. Ten blind samples of swine urine were analyzed simultaneously by icELISA, LFA, and ultra-high-performance liquid chromatography–tandem mass spectrometry, and the results of the three methods agreed well. Therefore, the combination of two immunoassays provides an effective and rapid screening method for detection of PA residues in biological samples. [Figure not available: see fulltext.] © 2015 Springer-Verlag Berlin Heidelberg


Li X.,China Agricultural University | Li X.,Beijing WDWK Biotechnology Company | Wang W.,Beijing WDWK Biotechnology Company | Wang L.,China Agricultural University | And 3 more authors.
Analytical and bioanalytical chemistry | Year: 2015

Phenylethanolamine A (PA) is a β-adrenergic agonist, which was first used in animal husbandry as a growth promoter in China in 2010. In this study, a monoclonal-antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) and lateral-flow immunoassay (LFA) for the detection of PA in swine urine and pork were developed. The immunogen was prepared by linking PA hapten with carrier protein via a diazotization method. The IC50 value of the optimized icELISA was 0.44 ng mL(-1). The limits of detection of the icELISA for PA in swine urine and pork were 0.13 ng mL(-1) and 0.39 ng g(-1), respectively. The recoveries of PA from spiked swine urine and pork were in the range 82.0-107.4 % and 81.8-113.3%, respectively, with the coefficients of variation in the range 4.1-16.2% and 1.2-6.3%, respectively. The mAbs had negligible cross reactivity with 10 other β-agonists. In contrast, the LFA had a cut-off level of 5 ng mL(-1) (g) in swine urine and pork, and the results could be achieved within 5 min. Ten blind samples of swine urine were analyzed simultaneously by icELISA, LFA, and ultra-high-performance liquid chromatography-tandem mass spectrometry, and the results of the three methods agreed well. Therefore, the combination of two immunoassays provides an effective and rapid screening method for detection of PA residues in biological samples.


Li X.,China Agricultural University | Li X.,Beijing WDWK Biotechnology Co. | Shen J.,China Agricultural University | Wang Q.,China Agricultural University | And 4 more authors.
Analytical and Bioanalytical Chemistry | Year: 2015

A rapid, reliable, sensitive, and quantitative multi-residue fluorescent microspheres immunochromatographic assay (FMCA) was developed for simultaneous detection of four macrolides in raw milk. The IC50 value of the optimized FMCA was 1.36, 1.22, 1.01, and 1.39 ng/mL for erythromycin (ERY), spiramycin (SPI), tilmicosin (TIM), and tylosin (TYL), respectively. The limits of detection (LODs) for the four macrolides was 0.13 ng/mL. The recoveries of ERY, SPI, TIM, and TYL from spiked raw milk ranged from 91.8-109.2, 89.6-114.4, 84.8-111.6, and 85.8-115.2 %, respectively, with coefficients of variation (CVs) of 5.4-11.3, 7.9-15.7, 6.2-13.7, and 3.2-14.9 %, respectively. The whole testing process was completed within 20 min. The antibody-mixed labeled method was successfully applied to the FMCA, which greatly simplified the operation steps and saved a lot of time. Compared with the immunogold chromatographic assay (IGCA), the FMCA is more sensitive and stable and has less antibody consumption. A parallel analysis in blind raw milk samples was conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS); the results showed good correlation (r 2 = 0.99) between the two methods. Therefore, the developed multi-residue FMCA is reliable and can be easily applied to other antibiotics or other contaminants. [Figure not available: see fulltext.] © 2015 Springer-Verlag Berlin Heidelberg.


Yu X.,China Agricultural University | Tao X.,Southwest University | Shen J.,China Agricultural University | Shen J.,National Reference Laboratory for Veterinary Drug Residues | And 7 more authors.
Analytical Methods | Year: 2015

A one-step generic chemiluminescence competitive direct enzyme immunoassay (CL-cdELISA) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein (CL-cdELISAscFv-AP) was developed. It was capable of detecting 20 targeted fluoroquinolones (FQs) in fish and shrimp matrices within 40 min below the maximum residue levels (MRLs). In the optimized generic assay, the scFv-AP fusion protein in combination with a norfloxacin-ovalbumin conjugate (NOR-OVA) showed 50% binding inhibition (IC50) at 0.15 ± 0.01 μg kg-1 for NOR in 0.01 M phosphate-buffered saline (PBS), indicating that it is seven times as sensitive as the corresponding competitive direct enzyme immunoassay (cdELISAscFv-AP), and the linear response range of the assay was extended from 0.04 to 1.08 μg kg-1. The limits of detection (LODs) of the assay for NOR were 0.017 μg kg-1 in shrimp and 0.018 μg kg-1 in fish, and the LODs inferred from the cross reactivity (CR) ranged from 0.013 μg kg-1 for ciprofloxacin (CIP) to 4.19 μg kg-1 for trovafloxacin (TRO); the recoveries of the three representative antibiotics norfloxacin, flumequine (FLU) and sarafloxacin (SAR) from spiked fish and shrimp samples varied from 72.50 to 118.50% and the mean coefficients of variation for the inter-assay and intra-assay were 6.4% and 9.2%, respectively. Further validation of CL-cdELISAscFv-AP with high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed that the assay was a reliable screening tool for the detection of FQs in fish and shrimp. © 2015 The Royal Society of Chemistry.


Zhu J.,China Agricultural University | Tao X.,China Agricultural University | Ding S.,China Agricultural University | Shen J.,China Agricultural University | And 7 more authors.
Analytical Letters | Year: 2012

Zeranol (α-Zearalanol, α-ZAL), well-known as an anabolic promoter, was officially banned in Europe due to its potential carcinogenic and endocrine-disrupting biological activities. In this study, a method for the determination of zeranol residues in bovine milk and urine was developed based on micro-plate chemiluminescence enzyme immunoassay (CLEIA). The limit of detection (LOD) was 50 ng/L, and the linear range was between 100 ng/L and 4510 ng/L, with a correlation coefficient (R2)0.9982. The recoveries of zeranol in bovine milk and urine were between 84.7% and 123.6%. This study showed that CLEIA was a reliable, convenient, and sensitive method for screening zeranol residues in bovine milk and urine. © 2012 Copyright Taylor and Francis Group, LLC.


PubMed | Beijing WDWK Biotechnology Co. and China Agricultural University
Type: Evaluation Studies | Journal: Analytical and bioanalytical chemistry | Year: 2016

A rapid, reliable, sensitive, and quantitative multi-residue fluorescent microspheres immunochromatographic assay (FMCA) was developed for simultaneous detection of four macrolides in raw milk. The IC50 value of the optimized FMCA was 1.36, 1.22, 1.01, and 1.39 ng/mL for erythromycin (ERY), spiramycin (SPI), tilmicosin (TIM), and tylosin (TYL), respectively. The limits of detection (LODs) for the four macrolides was 0.13 ng/mL. The recoveries of ERY, SPI, TIM, and TYL from spiked raw milk ranged from 91.8-109.2, 89.6-114.4, 84.8-111.6, and 85.8-115.2%, respectively, with coefficients of variation (CVs) of 5.4-11.3, 7.9-15.7, 6.2-13.7, and 3.2-14.9%, respectively. The whole testing process was completed within 20 min. The antibody-mixed labeled method was successfully applied to the FMCA, which greatly simplified the operation steps and saved a lot of time. Compared with the immunogold chromatographic assay (IGCA), the FMCA is more sensitive and stable and has less antibody consumption. A parallel analysis in blind raw milk samples was conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS); the results showed good correlation (r(2)=0.99) between the two methods. Therefore, the developed multi-residue FMCA is reliable and can be easily applied to other antibiotics or other contaminants.


PubMed | Beijing WDWK Biotechnology Company and China Agricultural University
Type: Evaluation Studies | Journal: Analytical and bioanalytical chemistry | Year: 2015

Phenylethanolamine A (PA) is a -adrenergic agonist, which was first used in animal husbandry as a growth promoter in China in 2010. In this study, a monoclonal-antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) and lateral-flow immunoassay (LFA) for the detection of PA in swine urine and pork were developed. The immunogen was prepared by linking PA hapten with carrier protein via a diazotization method. The IC50 value of the optimized icELISA was 0.44 ng mL(-1). The limits of detection of the icELISA for PA in swine urine and pork were 0.13 ng mL(-1) and 0.39 ng g(-1), respectively. The recoveries of PA from spiked swine urine and pork were in the range 82.0-107.4 % and 81.8-113.3%, respectively, with the coefficients of variation in the range 4.1-16.2% and 1.2-6.3%, respectively. The mAbs had negligible cross reactivity with 10 other -agonists. In contrast, the LFA had a cut-off level of 5 ng mL(-1) (g) in swine urine and pork, and the results could be achieved within 5 min. Ten blind samples of swine urine were analyzed simultaneously by icELISA, LFA, and ultra-high-performance liquid chromatography-tandem mass spectrometry, and the results of the three methods agreed well. Therefore, the combination of two immunoassays provides an effective and rapid screening method for detection of PA residues in biological samples.

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